Applied biosystems stepone plus real time pcr systems

RNA was isolated with an RNeasy Mini kit (Qiagen) and cDNA was generated by reverse transcription (Applied Biosystems). Real-time RT-PCR was performed with a SYBR green PCR mix (ABI Biosystems) in the Realplex Eppendorf Real-time PCR instrument (Eppendorf AG). Gene expression levels were calculated relative to the 18s gene. Data were collected and quantitatively analyzed on a Realplex sequence detection system (Eppendorf AG), and Applied Biosystems StepOne Plus Real-time PCR systems (Applied Biosystems). The primer sequences are listed in Supplementary Table 1.

RNA is extracted from cell pellets using the RNeasy Mini Kit (Qiagen, Hilden, Germany). Complementary cDNA is then synthesized using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA). qPCR is performed using the Applied Biosystems StepOnePlus Real-Time PCR systems (Thermo Fischer Scientific). Primer sets were validated with transient temperature PCR and Platinum SYBR Green qPCR Supermix-UDG (Thermo Fischer Scientific). Following primer pairs were selected for LC3 (MAP1LC3B) mRNA: 5′-GCGACTGGAGAGCTGTTTCT and 5′-AACCACATCCTAAGGCCAGC; for beta-ACTIN: 5′-ATGCTCCCCGGGCTGTAT and 5′-CATAGGAGTCCTTCTGACCATTC; and for GAPDH: 5′-TGTGTCCGTCGTGGATCTGA and 5′-CCTGCTTCACCACCTTCTTGA. (Taqman-probes were then created for LC3, beta-ACTIN (5′-CCTAGGCACCAGGGTGTGATG) and GAPDH (5′-CCGCCTGGAGAAACCTGCCAAGTATG) and combined with the PrimeTime Gene Expression Master Mix from Thermo Fischer Scientific. For other mRNA, we used primer and probe sets from Thermo Fischer Scientific: Mm00465434_m1 (mPKD1; spanning exon 1–2), Mm00435829_m1 (mPKD2; spanning exon 1–2), Mm01187303_m1 (mATG5), Mm00503201_m1 (mATG12), Mm01265461_m1 (mBECN1), Mm00448091_m1 (mSQSTM1) and mHif1a (Mm00468869_m1). qPCR was performed with the corresponding StepOnePlus Real-Time PCR System Software and analyzed with the 2^−ΔΔCt method [50 (link)].